Electron spin resonance spectroscopy (ESR) is among the most useful methods of detecting, measuring and studying paramagnetic species. The ESR spectrum is an absorption spectrum showing the energy required to change the spin state of an unpaired electron from a preferred alignment with an externally applied magnetic field to the less stable spin state alignment against the externally applied magnetic field. Similar to nuclear magnetic resonance spectra, the ESR spectra show signal splitting because of coupling of the spins of nearby nuclei with atoms that carry the unpaired electron.
ESR has been used spectroscopically to determine the presence and concentration of paramagnetic species in a test sample under analysis. Typically a very small test sample is subjected to a fixed radio frequency field in the so-called X band (e.g., 9.5 Gigahertz) and to a time-varying homogenous magnetic field. The resulting absorption spectrum is detected as a signal of the presence and concentration of a paramagnetic species within the test sample having electron spin resonances corresponding to magnetic field intensities that fall within the detected spectrum for the particular fixed radio frequency being used. Typically the magnetic field is on the order of several thousand gauss and is provided by placing the sample between the poles of an electromagnet. The time variation in the magnetic field is accomplished by varying the current in a pair of sweep coils positioned respectively between each pole of the electromagnet and the sample.
As a result, ESR spectroscopy can be used to detect paramagnetic species, like free radicals; to measure the concentration of paramagnetic species; and to provide information as to the structure of the paramagnetic species. Furthermore, ESR spectroscopic techniques are extremely sensitive, such that, under favorable conditions, a free radical concentration of as low as 10.sup.-12 M can be detected. Therefore, ESR spectroscopic techniques have been used to study free radical intermediates in organic reactions, to study the generation and decay of free radical species in oxidations catalyzed by enzymes, to detect drug metabolites, to detect quinones that are present in all biological species, to study the mechanism of drug or toxin interactions with cellular constitutents, and to study the fixing of carbon dioxide in algae during photosynthesis.
However, not all paramagnetic species are amenable to direct detection and measurement by ESR spectroscopic techniques. For example, molecular oxygen is a stable free radical having two unpaired electrons. However, the direct measurement of the concentration of molecular oxygen, especially in aqueous solution, by ESR spectroscopic techniques is not possible because the ESR absorption signal is too broad. The extreme breadth of the ESR absorption signal for molecular oxygen is due to the existence of two intramolecular spins and four intramolecular spin states that have extremely short-lived spin states, and that are in an environment of frequent molecular collisions. Since the breadth of the spectral signal, .GAMMA.,is equal to the inverse of the average lifetime of the spin (T.sub.1), the very small T.sub.1 for molecular oxygen, especially in aqueous solution, makes the breadth of the spectral signal immeasurably large.
Molecular oxygen, however, can be detected and measured, even in aqueous solution, through an ESR spectroscopic technique that determines the effect of molecular oxygen on the lifetimes of the longer-lived spin states of other paramagnetic compounds. The longer-lived spin states are found in paramagnetic organic molecules having a stable unpaired electron. Such paramagnetic organic molecules are observable by ESR spectroscopy and commonly have narrow, well-measured hyperfine spectra that are detectably broadened in the presence of molecular oxygen. The broadening of the hyperfine spectra is essentially directly proportional to the molecular oxygen concentration in the test sample, and therefore the degree of spectrum broadening is used to quantify the amount of molecular oxygen in the test sample.
The broadening of the ESR spectrum occurs because of an interaction between the spin of the free electron present on the paramagnetic organic molecule and the spins of the unpaired electrons of the molecular oxygen. The exchange of spins between the paramagnetic organic molecule and the molecular oxygen allows the relatively long-lived spin state of the paramagnetic organic molecule to couple with the extremely short-lived spin states of molecular oxygen, thereby short-circuiting the normal spin relaxation mechanisms of molecular oxygen. The resulting shortened lifetime of the spin state of the paramagnetic organic molecule is reflected in a broadening of the narrow spectral line of the paramagnetic organic molecule. In this ESR spectroscopic technique, the amount of the paramagnetic organic molecule added to the test sample need only be high enough to generate a detectable ESR spectrum and to permit measurement of oxygen-induced spectrum broadening. Accordingly, the sharpest, narrowest ESR spectra correspond to the lowest molecular oxygen concentrations.
The paramagnetic organic molecules having an unpaired electron and used to help detect, measure and monitor the concentration of molecular oxygen, organic-free radicals and other paramagnetic species in a test sample are termed spin labels. Therefore, in order to determine the oxygen concentration, or oxygen tension, of a test sample by ESR spectroscopic techniques, a spin label is added to the test sample to act as a reporter substance. Consequently, a suitable spin label should be capable of incorporation into the test sample and capable of reporting information concerning the paramagnetic species of interest to a detector, such as an ESR spectrometer; should have paramagnetic properties different from the paramagnetic properties of the paramagnetic species of interest in the test sample; and should not interact with the test sample, physically or chemically, to cause measurable changes in the test sample.
The most advantageous spin labels are stable paramagnetic organic molecules having a free unpaired electron and of low reactivity. Although a variety of inorganic spin labels have been employed, such as nitric oxide, paramagnetic transition metal ions and lanthanide ions, the most useful spin labels, both in solution and in biological samples, including living organisms, have been organic spin labels, especially the protected nitroxide compounds of general structural formula (I) that include the paramagnetic nitroxyl moiety. ##STR1## The nitroxides of general structural formula I have been widely used as spin labels because they are extremely stable, and extremely inert, due to the protective effect of the four methyl groups substituted on the alpha and alpha' carbon atoms. The stability of the free radical portion of the molecule of general structure I, i.e., the nitroxyl moiety, allows a variety of chemical reactions to be performed on the A portion of the molecule in order to alter the physical and chemical characteristics of the nitroxide spin label molecule without destroying the free radical portion of the molecule. Similarly, the stability of the free radical nitroxyl portion of the nitroxide allows the nitroxyl moiety to remain unchanged in biological samples, therefore allowing the study of free radical and paramagnetic species both in vitro and in vivo. It also should be noted that the four methyl groups substituted on the alpha and alpha' carbon atoms help stabilize the nitroxide molecule by making a disproportionation reaction between two nitroxide molecules to yield a nitrone and a hydroxyamine, and therefore destroy the paramagnetism of the molecule, more difficult.
The nitroxides of structural formula I have proven very useful in ESR spectroscopic techniques to measure molecular oxygen tension, or molecular oxygen concentration, in aqueous solution. For example, it is well known that the interaction of dissolved oxygen molecules and nitroxide free radicals, through Heisenberg spin-exchange, causes broadening of the ESR spectral lines. Therefore, with a suitable nitroxide spin label, this property can be used to quantitate oxygen concentrations in solution. In addition, the nitroxides do not consume oxygen, thereby allowing the detection and measurement of slow-rate oxygen consumption processes over a long period of time. Consequently, the nitroxide spin label CTPO has been used to monitor the concentration of dissolved oxygen in the aqueous regions of biological samples.
The effect of molecular oxygen on the width of an ESR spectra of a nitroxide spin label was originally reported by G. F. Pake and T. R. Tuttle, Jr. in the publication, nomalous Loss of Resolution of "A Paramagnetic Resonance Hyperfine Structure in Liquids", Physical Review Letters, 3(9):423 (1959), wherein the authors theorized that the spectrum widening effect due to molecular oxygen was an anomaly. However, it was this initially-theorized anomaly that eventually led to the T.sub.2 -based, or spectrum width, spin label oxymetry method of determining the molecular oxygen concentration of a test sample. M. J. Popovitch, in the publication "Electron Spin Resonance Oxygen Broadening", J. Phys. Chem. 79 (11): 1106-1109 (1975) and J. M. Backer, J. M. Budker, S. L. Eremenko, and Yu N. Molin, in the publication "Detection of the Kinetics of Biochemical Reactions with Oxygen Using Exchange Broadening in the ESR Spectra of Nitroxide Radicals", Biochemica et Biophysica Acta 460:152-156 (1977) used the spectra broadening effect of molecular oxygen on the ESR spectrum of a nitroxide spin label in a biological sample to make indirect measurements of the ambient oxygen tension. Popovitch and Backer each utilized nitroxide compounds as the spin labels, particularly nitroxide nitrogen-heterocycles having di-methyl substitutions on the alpha and the alpha' carbon atoms, as depicted in structural formula I. These small nitroxide spin label molecules rotated sufficiently rapidly in solution, and in the hydration shells of a biological environment, to average out hyperfine and coupling pseudotensor anisotropies, therefore yielding, in the absence of oxygen, narrow, well-defined hydrogen hyperfine spectra. However, with the addition of oxygen to the test sample, these narrow hydrogen hyperfine splittings, in a roughly proportionate fashion, broaden and disappear. Oxymetry, or oxygen measurements, performed by this ESR method utilize the broadening effect of oxygen concentration on the linewidth of the proton hyperfine lines of the ESR spectra and, therefore, are termed T.sub.2 -based.
However, the use of a nitroxide spin label in an ESR method to measure the oxygen tension of a test sample still possesses several disadvantages. To date, two approaches have been used for T.sub.2 -based ESR spin label oxymetry, each having advantages and disadvantages. The first approach is the standard nitroxide spin label ESR oxymetry method as described by Lai, et al., in the publication "ESR Studies of O.sub.2 Uptake By Chinese Hamster Ovary Cells during the Cell Cycle" Proc.Natl.Acad. Sci. USA 79:1166-1170 (1982) and by the previously cited publication of Backer et al. The ESR spectrum of an aqueous solution of the nitroxide spin label 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxyl (CTPO) shows a broadening of the proton hyperfine lines upon the addition of oxygen, such that the height of the hyperfine lines relative to the nitrogen triplet is reduced roughly proportionately to the oxygen tension. In this technique, a ratio between the height of a proton hyperfine line and the overall height of the spectrum of the nitrogen line under consideration is found, and the oxygen tension determined. In this standard oxymetry method, the ratio used to determine oxygen tension is independent of the amount of nitroxide spin label present in the test sample, except for normal signal-to-noise limitations. According to this standard method, the ESR data pertaining to the measurement of the oxygen tension is related to the spectral shape and is independent of the spectral height.
The above-described standard method suffers a serious disadvantage because the very narrow hyperfine lines, as a principal source of the width of the nitroxide signal, are dispersed over the full width of the nitroxide signal. Furthermore, these hyperfine lines are resolved only by reduction of the ESR spectrometer field modulation amplitude, thereby reducing the intensity of the spectral signal. As a result, only a relatively small fraction of the nitroxide spin label is contributing to the signal at any one field point therefore yielding relatively low signal intensity.
A second ESR oxymetry method that has been investigated uses a perdeuterated nitroxide spin label. The hyperfine coupling of the deuterium to the unpaired electron of the nitroxide is roughly one-seventh the hyperfine coupling of hydrogen. Therefore, the deuterium hyperfine lines are so closely spaced that the hyperfine lines are barely resolvable by ESR spectrometers. Therefore, the resulting ESR spectrum is a narrow, easily measured single line. In contrast to the ESR spectrum of the standard, hydrogenated nitroxide spin label described above, measurements from ESR spectra of the perdeuterated nitroxide spin label are easier to perform because all of the perdeuterated nitroxide spin label is contributing to the signal over a very narrow region. Furthermore, it has been shown that the width of this single line signal is very sensitive to, and is roughly proportional to, the ambient oxygen tension. As a result, the height of the narrow ESR spectrum of a deuterated nitroxide spin label varies dramatically with the oxygen tension because at a fixed, small modulation field amplitude the height of a line described by the first derivative of a Lorentzian shape is proportional to the inverse square of the width of the line.
However, the perdeuterated nitroxide spin labels also present disadvantages when used in ESR oxymetry methods. For example, unlike the hydrogenated nitroxide spin labels, the height of the perdeuterated nitroxide spin label spectrum is sensitive both to the oxygen tension of the test sample and to the concentration of spin label in the test sample. In addition, the dependence of the perdeuterated spin label signal height upon perdeuterated spin label concentration, at constant oxygen tension, is not linear over the entire range of spin label concentration. The linear increase in signal height with increasing concentration of perdeuterated spin label, at low concentrations of perdeuterated spin label, is eventually overcome by the self-broadening effect of the perdeuterated spin label at higher concentrations of perdeuterated spin label, therefore yielding a linear decrease of the signal height at higher perdeuterated spin label concentrations.
If a test sample has a uniform perdeuterated spin label distribution, data relating to the amount of perdeuterated spin label contributing to the ESR spectrum can be determined from the double integral of the derivative ESR spectrum. However, the sensitivity of the oxygen tension measurement will be reduced by the uncertainties inherent in that calculation. In addition, in a sample having a heterogeneous distribution of the perdeuterated spin label, such as in biological samples, the contribution of the spin label concentration to the spectrum is even more difficult to determine, as is the interpretation of the spin label concentration-weighted spectral width. Therefore, given the sensitivity of the spectral width to perdeuterated spin label concentration, and the difficulty of reliably determining the contribution of perdeuterated spin label concentrations from heterogeneous samples, a reliable and accurate quantitative measurement of oxygen tension from actively metabolizing heterogeneous tissue is difficult. Furthermore, perdeuterated nitroxide spin labels may contain labile, or exchangeable, hydrogen/denterium atoms on functional groups at sites that effect the spectral linewidth. Therefore, in a method to determine the oxygen tension in live organisms, the deuterium present at these labile sites would be exchanged for hydrogen, and thereby render the oxygen tension measurement unreliable.
Accordingly, to date, the compounds used as spin labels in ESR studies of paramagnetic species have suffered from sacrificing one beneficial property in order to achieve another beneficial property. Prior to the present invention, no known class of compounds has effectively provided a strong, narrow signal that is essentially independent of nitroxide spin label concentration in the test sample. Therefore, it would be advantageous to have a spin label for use in an ESR-based assay method, such as oxymetry, that provides a strong, narrow spectral line that eliminates much of the usual signal-to-noise problem, as demonstrated by the perdeuterated nitroxide spin labels, and also provides an ESR spectrum that is essentially independent of the amount of nitroxide spin label present in the test sample, as demonstrated by the standard, hydrogenated nitroxide spin labels. Surprisingly and unexpectedly, the new class of compounds of the present invention, the selectively isotopically-labeled spin labels, provides the advantages, and avoids the disadvantages, of both the standard hydrogenated nitroxide spin labels and the perdeuterated nitroxide spin labels.
Therefore, in accordance with the present invention, a new class of compounds is used in an improved method of assaying a test sample, such as a solution or a living organism, for the presence and concentration of a paramagnetic species, such as molecular oxygen, through ESR spectroscopic techniques. The new class of spin label compounds include selectively isotopically-labeled nitroxide spin labels, wherein at least one, but less than all, of the non-labile hydrogen atoms have been replaced by deuterium atoms, or wherein an isotope of a particular atom replaces at least one, but not all, of the particular atoms present in the molecule. In addition, the new class of compounds sufficiently enhance the sensitivity of ESR spectroscopic techniques, such that low frequency ESR spectrometers, that yield weak signals, can be used in assays to detect, measure and monitor paramagnetic species in a test sample.